Mechanism Of Manganese Regulation Of CGAS-STING In Immunity To Mycobacterium Tuberculosis Infection

Objective:To investigate the essential role and intrinsic mechanism of Mn²⁺in host cell resistance to Mycobacterium tuberculosis（M.tb）,provide further insight into the interaction between M.tb and the host,contribute to the use of Mn²⁺as a drug cofactor in the treatment of tuberculosis（TB）and the development of a new generation of vaccine adjuvants.It also provided theoretical support for the development of a new generation of vaccine adjuvants.Methods:The survival of M.tb H37Ra in macrophages,both intracellular and extracellular,was detected by dilution coating.The integrity of the cell membrane of macrophages was detected by nuclear skeleton staining.The phosphorylation level of the necroptosis-executing protein MLKL in macrophages was detected by immunofluorescence.A mouse model infected with M.tb H37Ra was established and the infiltration of inflammatory cells in the lung and spleen tissues of mice was observed by HE staining.Immunofluorescence was used to detect the transfer of p P65 inside and outside the nucleus.ELISA to detect changes in the ability of macrophages to secrete TNF-α,and qPCR to detect the gene expression of TNF-αin macrophages.The key proteins of the STING pathway,western blot was used to detect phosphorylation levels of STING and P65 in macrophages.And qPCR to detect the expression of STING and c GAS genes macrophages.The effect of Mn²⁺in macrophages was simulated using c GAMP,an activator of STING protein.Transcriptome analysis of Mn²⁺-treated/untreated macrophages infected with M.tb H37Ra was performed.The phosphorylation levels of the TNF pathway-related proteins ERK,P38 and JNK obtained from transcriptome analysis were examined by Western Blot.qPCR was performed to detect the expression of TNF pathway-related genes CXCL10,CCL20,CSF1,CSF2 and JAG1 H-151 and the expression of TNF signaling pathway-related proteins and genes after knockdown of STING protein by si RNA.Results:In this study,we found that Mn²⁺significantly inhibited the viability of intracellular bacteria in macrophages infected with M.tb H37Ra,but had no significant effect on the viability of extracellular bacteria,and that Mn²⁺induced necroptosis in macrophages.In HE-stained sections of lung and spleen tissues from mice infected with M.tb H37Ra,Mn²⁺enhanced inflammatory cell infiltration around the lung and spleen;in a cellular model,Mn²⁺significantly increased the ability of macrophages infected with M.tb H37Ra to secrete TNF-α,and increased levels of P65 phosphorylation showed an extra-nuclear to intra-nuclear shift.Mn²⁺significantly increased the phosphorylation levels of STING and P65 compared with those of macrophages infected with M.tb H37Ra,thereby activating the STING pathway.RNA-Seq showed significant enrichment of the TNF signalling pathway,with significant differences in CXCL10,CCL20,CSF1,CSF2 and JAG1 genes,and significant phosphorylation of the TNF pathway-related proteins JNK,ERK and P38.H-151 and small interfering RNA knockdown of STING resulted in a corresponding down-regulation of TNF pathway-related gene expression and protein phosphorylation levels.Conclusion:1.Mn²⁺regulated the production of TNF-αby M.tb-infected macrophages through the STING-TNF pathway and induced necroptosis in macrophages.2.Mn²⁺inhibited the growth of bacteria in macrophages infected with M.tb showing great application prospect in the treatment of TB.